Project Summary Chronic myeloid leukemia (CML) is a lethal malignancy. Today, tyrosine kinase inhibitors (TKIs) effectively treat the disease, leading to rapid expansion of long-term CML survivors. However, TKIs fail to eradicate CML leukemia stem cells (LSCs) and the disease relapses when the drug is stopped. As a result, CML patients need life-long dependence on TKIs. There is a need to eradicate CML LSCs for a cure. Our long-term goal is to understand the mechanisms of CML LSC drug resistance, and to develop novel strategies to eradicate CML LSCs and improve CML treatment. We have previously shown that protein deacetylase SIRT1 is activated by BCR-ABL transformation and promotes CML progression and LSC drug resistance. In our recent study of hematopoietic stem cell (HSC) aging, we identified an E3 ubiquitin ligase Trim26 as a novel Sirt1 effector in old mouse HSCs. We found that Trim26 inhibition had strikingly similar effect to SIRT1 inhibition on suppressing CML cell growth and survival. Trim26 was over-expressed in both human and mouse CML progenitor cells, and Trim26 knockout reduced CML LSCs. Unlike Sirt1 knockout, Trim26 knockout did not affect normal mouse development and functions. The objective of this application is to determine the roles of Trim26 in regulating CML LSC survival and self-renewal. Our central hypothesis is that Trim26 promotes CML LSC survival and maintenance and facilitates leukemia development. The rationale for the proposed research is that better understanding the roles of Trim26 in CML LSCs will help design a more effective and safer strategy to eradicate CML LSCs and bring a cure to the disease. We will test our central hypothesis in two specific aims: 1) To determine the role of Trim26 in CML LSC survival and self-renewal; 2) To determine the mechanisms of Trim26 in regulating CML LSCs. Under aim 1, the effect of Trim26 knockout on CML LSC survival and maintenance as well as the LSC persistence upon TKI treatment will be determined using a mouse model of CML. Under aim 2, the requirement of Trim26 E3 ubiquitin ligase activity for LSC functions and CML development, and novel ubiquitination substrates of Trim26 will be determined for regulation of CML LSC functions and leukemia development. The proposed research is significant because it will reveal a new mechanism of CML LSC survival and drug resistance, and identify Trim26 as a novel and safe therapeutic target to eradicate CML LSCs.